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Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain

View Article: PubMed Central - PubMed

ABSTRACT

The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska complex binds dynamic microtubules using multiple contact sites in a mode that allows conformation-independent binding. Here, we show that the Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex (i) by directly interacting with tubulin monomers and (ii) indirectly by interacting with tubulin contacting regions of Ska1 suggesting an allosteric regulation. Perturbing either the Ska3-microtubule interaction or the Ska3-Ska1 interactions negatively influences microtubule binding by the Ska complex in vitro and affects the timely onset of anaphase in cells. Thus, Ska3 employs additional modulatory elements within the Ska complex to ensure robust kinetochore-microtubule attachments and timely progression of mitosis.

No MeSH data available.


The intrinsically disordered C-terminal domain of Ska3 contributes to the microtubule binding activity of the Ska complex.(a) Domain architecture of the Ska components where filled boxes represent structured regions. MTBD: MicroTubule Binding Domain of Ska1 (residues from 133 to 255). (b) Predicted disordered and protein-binding regions of Ska3 using Disopred (http://bioinf.cs.ucl.ac.uk/psipred). Blue: predicted disordered residues, Orange: predicted protein-binding residues. (c) Amino acid conservation of Ska3 (conservation score is mapped from red to cyan, where red corresponds to highly conserved and cyan to poorly conserved). The alignments include orthologs from H. sapiens (hs), Bos taurus (bt), Sus scrofa (ss), Mus musculus (mm), Echinops telfairi (et), Orcinus orca (oo). Ska3 residues evaluated in this study are marked with asterisks. Multiple sequence alignment was performed with MUSCLE (MUltiple Sequence Comparison by Log-Expectation, EMBL-EBI) and edited with Aline35. (d) Quantification of MT-cosedimentation assay comparing the microtubule-binding activity of the wt Ska complex, Ska1ΔC and Ska3ΔC. Concentrations used in the assay: 1 μM protein, 9 μM MTs (mean ± s.d., n = 3, **P ≤0.01, ***P ≤0.001; t-test). (e) Left, Representative SDS-PAGE of MT-cosedimentation assays comparing the microtubule-binding activity of the wt Ska complex, Ska1ΔC and Ska3ΔC. Right, Microtubule-binding curve for the wt Ska complex, Ska1ΔC and Ska3ΔC. Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n = 4).
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f1: The intrinsically disordered C-terminal domain of Ska3 contributes to the microtubule binding activity of the Ska complex.(a) Domain architecture of the Ska components where filled boxes represent structured regions. MTBD: MicroTubule Binding Domain of Ska1 (residues from 133 to 255). (b) Predicted disordered and protein-binding regions of Ska3 using Disopred (http://bioinf.cs.ucl.ac.uk/psipred). Blue: predicted disordered residues, Orange: predicted protein-binding residues. (c) Amino acid conservation of Ska3 (conservation score is mapped from red to cyan, where red corresponds to highly conserved and cyan to poorly conserved). The alignments include orthologs from H. sapiens (hs), Bos taurus (bt), Sus scrofa (ss), Mus musculus (mm), Echinops telfairi (et), Orcinus orca (oo). Ska3 residues evaluated in this study are marked with asterisks. Multiple sequence alignment was performed with MUSCLE (MUltiple Sequence Comparison by Log-Expectation, EMBL-EBI) and edited with Aline35. (d) Quantification of MT-cosedimentation assay comparing the microtubule-binding activity of the wt Ska complex, Ska1ΔC and Ska3ΔC. Concentrations used in the assay: 1 μM protein, 9 μM MTs (mean ± s.d., n = 3, **P ≤0.01, ***P ≤0.001; t-test). (e) Left, Representative SDS-PAGE of MT-cosedimentation assays comparing the microtubule-binding activity of the wt Ska complex, Ska1ΔC and Ska3ΔC. Right, Microtubule-binding curve for the wt Ska complex, Ska1ΔC and Ska3ΔC. Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n = 4).

Mentions: Amino acid sequence analysis of Ska3, using secondary structure and disorder prediction methods, revealed the presence of an intrinsically disordered C-terminal domain of unknown function (amino acids 102–412, Ska3C-term; Fig. 1a,b), with high propensity to participate in protein-protein interactions (Fig. 1b). This domain is highly acidic in nature (theoretical isoelectric point is ~4.5) and possesses a high proportion of prolines (30 out of 311 residues; 9.6%), features that are remarkably conserved among species (Fig. 1c). Our previous work showed that the Ska3C-term, although did not bind microtubules on its own, when removed from the Ska complex reduced the microtubule ability of the latter24. This had suggested that Ska3C-term might favorably contribute to the microtubule binding of the Ska complex by creating microtubule-binding interfaces, together with Ska1, when connected to the structural core of the Ska complex made of coiled-coils24. To quantitatively assess this possibility, we carried out microtubule cosedimentation assays with Ska complexes lacking the C-terminal domain of Ska1 (Ska11–91-Ska2-Ska3; Ska1ΔC) or the C-terminal domain of Ska3 (Ska1-Ska2-Ska31–101; Ska3ΔC). While the Ska3ΔC complex showed reduced microtubule binding, the Ska1ΔC complex, as expected24, failed to interact with microtubules (Fig. 1d and e, Supplementary Fig. S1a). Microtubule binding affinity of the Ska3ΔC complex, as measured by the microtubule cosedimentation assay, is 13.6 μM, which is 4.5 fold lower than that of wild type (wt) Ska complex comprising full length Ska3 (Kd = 3 μM) (Fig. 1e). The analysis of the measured molecular mass of wt Ska complex and Ska3ΔC, determined by SEC-MALS (Size Exclusion Chromatography combined with Multi-Angle Light Scattering), showed that the removal of the Ska3C-term did not alter the oligomeric state of the complex (Supplementary Fig. S1b). Therefore, the reduced microtubule-binding of the Ska3ΔC complex is a direct consequence of the loss of the C-terminal domain of Ska3.


Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain
The intrinsically disordered C-terminal domain of Ska3 contributes to the microtubule binding activity of the Ska complex.(a) Domain architecture of the Ska components where filled boxes represent structured regions. MTBD: MicroTubule Binding Domain of Ska1 (residues from 133 to 255). (b) Predicted disordered and protein-binding regions of Ska3 using Disopred (http://bioinf.cs.ucl.ac.uk/psipred). Blue: predicted disordered residues, Orange: predicted protein-binding residues. (c) Amino acid conservation of Ska3 (conservation score is mapped from red to cyan, where red corresponds to highly conserved and cyan to poorly conserved). The alignments include orthologs from H. sapiens (hs), Bos taurus (bt), Sus scrofa (ss), Mus musculus (mm), Echinops telfairi (et), Orcinus orca (oo). Ska3 residues evaluated in this study are marked with asterisks. Multiple sequence alignment was performed with MUSCLE (MUltiple Sequence Comparison by Log-Expectation, EMBL-EBI) and edited with Aline35. (d) Quantification of MT-cosedimentation assay comparing the microtubule-binding activity of the wt Ska complex, Ska1ΔC and Ska3ΔC. Concentrations used in the assay: 1 μM protein, 9 μM MTs (mean ± s.d., n = 3, **P ≤0.01, ***P ≤0.001; t-test). (e) Left, Representative SDS-PAGE of MT-cosedimentation assays comparing the microtubule-binding activity of the wt Ska complex, Ska1ΔC and Ska3ΔC. Right, Microtubule-binding curve for the wt Ska complex, Ska1ΔC and Ska3ΔC. Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n = 4).
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f1: The intrinsically disordered C-terminal domain of Ska3 contributes to the microtubule binding activity of the Ska complex.(a) Domain architecture of the Ska components where filled boxes represent structured regions. MTBD: MicroTubule Binding Domain of Ska1 (residues from 133 to 255). (b) Predicted disordered and protein-binding regions of Ska3 using Disopred (http://bioinf.cs.ucl.ac.uk/psipred). Blue: predicted disordered residues, Orange: predicted protein-binding residues. (c) Amino acid conservation of Ska3 (conservation score is mapped from red to cyan, where red corresponds to highly conserved and cyan to poorly conserved). The alignments include orthologs from H. sapiens (hs), Bos taurus (bt), Sus scrofa (ss), Mus musculus (mm), Echinops telfairi (et), Orcinus orca (oo). Ska3 residues evaluated in this study are marked with asterisks. Multiple sequence alignment was performed with MUSCLE (MUltiple Sequence Comparison by Log-Expectation, EMBL-EBI) and edited with Aline35. (d) Quantification of MT-cosedimentation assay comparing the microtubule-binding activity of the wt Ska complex, Ska1ΔC and Ska3ΔC. Concentrations used in the assay: 1 μM protein, 9 μM MTs (mean ± s.d., n = 3, **P ≤0.01, ***P ≤0.001; t-test). (e) Left, Representative SDS-PAGE of MT-cosedimentation assays comparing the microtubule-binding activity of the wt Ska complex, Ska1ΔC and Ska3ΔC. Right, Microtubule-binding curve for the wt Ska complex, Ska1ΔC and Ska3ΔC. Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n = 4).
Mentions: Amino acid sequence analysis of Ska3, using secondary structure and disorder prediction methods, revealed the presence of an intrinsically disordered C-terminal domain of unknown function (amino acids 102–412, Ska3C-term; Fig. 1a,b), with high propensity to participate in protein-protein interactions (Fig. 1b). This domain is highly acidic in nature (theoretical isoelectric point is ~4.5) and possesses a high proportion of prolines (30 out of 311 residues; 9.6%), features that are remarkably conserved among species (Fig. 1c). Our previous work showed that the Ska3C-term, although did not bind microtubules on its own, when removed from the Ska complex reduced the microtubule ability of the latter24. This had suggested that Ska3C-term might favorably contribute to the microtubule binding of the Ska complex by creating microtubule-binding interfaces, together with Ska1, when connected to the structural core of the Ska complex made of coiled-coils24. To quantitatively assess this possibility, we carried out microtubule cosedimentation assays with Ska complexes lacking the C-terminal domain of Ska1 (Ska11–91-Ska2-Ska3; Ska1ΔC) or the C-terminal domain of Ska3 (Ska1-Ska2-Ska31–101; Ska3ΔC). While the Ska3ΔC complex showed reduced microtubule binding, the Ska1ΔC complex, as expected24, failed to interact with microtubules (Fig. 1d and e, Supplementary Fig. S1a). Microtubule binding affinity of the Ska3ΔC complex, as measured by the microtubule cosedimentation assay, is 13.6 μM, which is 4.5 fold lower than that of wild type (wt) Ska complex comprising full length Ska3 (Kd = 3 μM) (Fig. 1e). The analysis of the measured molecular mass of wt Ska complex and Ska3ΔC, determined by SEC-MALS (Size Exclusion Chromatography combined with Multi-Angle Light Scattering), showed that the removal of the Ska3C-term did not alter the oligomeric state of the complex (Supplementary Fig. S1b). Therefore, the reduced microtubule-binding of the Ska3ΔC complex is a direct consequence of the loss of the C-terminal domain of Ska3.

View Article: PubMed Central - PubMed

ABSTRACT

The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska complex binds dynamic microtubules using multiple contact sites in a mode that allows conformation-independent binding. Here, we show that the Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex (i) by directly interacting with tubulin monomers and (ii) indirectly by interacting with tubulin contacting regions of Ska1 suggesting an allosteric regulation. Perturbing either the Ska3-microtubule interaction or the Ska3-Ska1 interactions negatively influences microtubule binding by the Ska complex in vitro and affects the timely onset of anaphase in cells. Thus, Ska3 employs additional modulatory elements within the Ska complex to ensure robust kinetochore-microtubule attachments and timely progression of mitosis.

No MeSH data available.